Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo

BackgroundThe human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. MethodsExpression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/− flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted. ResultsExpression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice. ConclusionsIntestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.

背景：人类结肠内定植有大量细菌，已知此类细菌可广泛影响黏膜代谢与功能，且被认为参与炎症性肠病的发病机制及结肠癌的发生发展。本研究旨在探讨结肠细菌在体外及体内对上皮细胞分化相关因子的影响。我们以关键转录因子为研究重点：Hes1可引导细胞向吸收细胞谱系分化，Hath1与KLF4则调控杯状细胞的分化。
方法：将LS174T细胞与多种热灭活大肠杆菌（E. coli）菌株（包括益生菌大肠杆菌Nissle 1917，设置添加/不添加鞭毛蛋白的实验组）、乳杆菌及双歧杆菌共培养后，采用实时荧光定量PCR（real-time PCR）检测转录因子Hes1、Hath1、KLF4，黏蛋白Muc1、Muc2以及防御素HBD2的表达水平。蛋白层面的检测则通过蛋白质印迹（Western blot）实验及腔室玻片免疫染色完成。最后，分别检测无菌小鼠、无特定病原体（SPF）小鼠及常规定植小鼠结肠组织中上述因子的mRNA与蛋白表达水平，并对结肠杯状细胞进行计数。
结果：大肠杆菌K-12与大肠杆菌Nissle 1917可降低Hes1、Hath1的表达，对KLF4的表达亦存在轻微抑制作用。与之相反，Muc1与HBD2的表达显著上调，且该调控过程不依赖于Notch信号通路。益生菌大肠杆菌Nissle 1917通过鞭毛蛋白调控Hes1、Hath1、Muc1及HBD2的表达。体内实验验证了体外观察到的细菌作用：与无菌小鼠相比，无特定病原体小鼠及常规定植小鼠的结肠组织中Hath1与KLF4的表达水平降低，而结肠杯状细胞的数量无明显变化。
结论：肠道细菌可在体外及体内影响肠道上皮分化因子Hes1、Hath1、KLF4，以及黏蛋白Muc1与防御素HBD2的表达。Muc1与HBD2的诱导表达似乎直接由细菌触发，而非通过Notch信号通路介导。